Compounds for treatment of cell proliferative diseases

ABSTRACT

The present invention concerns compounds and their use to treat cell proliferative diseases such as cancer. Compounds of the present invention display significant potency as kinase inhibitors, cause the downregulation of c-myc, and inhibit the growth and survival of cancerous cell lines.

This application is a divisional of application Ser. No. 12/824,901,filed Jun. 28, 2010, now U.S. Pat. No. 8,119,827 which is a continuationof application Ser. No. 11/010,834, filed Dec. 13, 2004, now U.S. Pat.No. 7,745,468, which claims the benefit of U.S. Provisional ApplicationSer. No. 60/528,877 filed Dec. 11, 2003, the entire disclosure of whichis specifically incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the treatment of cellproliferative diseases such as cancer. More particularly, it concernstyrphostin and tyrphostin-like compounds useful for the treatment ofcell proliferative diseases such as cancer, methods of synthesis ofthese compounds, and methods of treatment employing these compounds.

2. Description of Related Art

AG490 is a kinase inhibitor that inhibits Jak2/Stat3 signaling. Targetedinhibition of the Jak/Stat pathway with AG490 inhibits tumor cell growthand increases sensitivity to apoptotic stimuli; thus, inhibitors of thispathway likely represent potential therapeutics for cancer therapy(Catlett-Falcone et al., 1999; Alas and Bonavida, 2003; Burdelya et al.,2002). Because IL-6 promotes survival and proliferation of certaincancerous cell lines through the phosphorylation of STAT3 (Bharti etal., Verma et al., Kerr et al.), kinase inhibitors similar to AG490 havepotential as anti-cancer drugs.

AG490 is structurally classified as a tyrphostin. U.S. Pat. No.6,596,828B2 and U.S. patent application 2003/0013748 describe compoundsthat have structural similarity with AG490.

Unfortunately, AG490 has limited activity in animal studies and must beused at high concentrations (˜50 to 100 μM) to achieve inhibition ofJak2/Stat3 signaling and anti-tumor effects, and this low potency ofAG490 is insufficient to warrant clinical investigation of this compoundfor the treatment of cancer (Burdelya et al., 2002; Meydan et al., 1996;Constantin et al., 1998). Thus a need exists for therapeutics thatexhibit strong anti-proliferative effects through a similar mechanism atlower therapeutic concentrations.

SUMMARY OF THE INVENTION

The present invention overcomes limitations in the art by providingcompounds that display improved pharmacological profiles (e.g.,increased potency) when compared with AG490; these compounds block IL-6mediated Stat3 activation at low concentrations (˜1 μM) and rapidlysuppress expression of the c-myc proto-oncogene, which is frequentlyoverexpressed, rearranged, or mutated in many malignancies (Hallek etal., 1998; Selvanayagam et al., 1988; Jernberg-Wiklund et al., 1992;Kuehl et al., 1997). Additionally, compounds of the present inventionalso induce apoptosis in c-myc overexpressing tumor cells that parallelstheir c-myc downregulatory activity. The present invention involvescompounds that have utility as antitumor and/or chemotherapeutic drugs,methods of synthesizing these compounds, and methods of using thesecompounds to treat patients with cancer.

One aspect of the present invention relates to a compound comprising thechemical formula:

wherein R₀ is selected from the group consisting of R₁ and R₁—Z₁—; and

wherein Z₁ is alkyl; and

wherein R₁ is chosen from the group consisting of:

where X₁, X₂, X₃, and X₄, are each independently selected from the groupconsisting of hydrogen, halogen, alkyl, alkoxy, OH, trihalomethyl, andNO₂;

where Y₁ is selected from the group consisting of halogen and O₂N; and

R₂ is selected from the group consisting of alkyl, alkenyl, alkynyl,alkoxy, alkylaryl, halogen, hydrogen, OH, NO₂, thioether, amine, SH, andNH₂;

R₃ is selected from the group consisting of:

wherein Z₃ is alkyl; and

wherein m₁=1, 2, 3, or 4; and

where R₄ is chosen from the group consisting of: CN, substituted amine,CH₂S-alkyl, alkyl, and CH₂N₃;

where R₅ and R₆ are each independently chosen from the group consistingof:

monosaccharide, monosaccharide derivative, polysaccharide,polysaccharide derivative, aryl, and alkylaryl;

where Z is selected from the group consisting of NH, S, and O, and

where X₅ and X₆ are each independently chosen from the group consistingof hydrogen and lower alkyl.

In certain embodiments of the present invention, R₄ is CN. In certainembodiments, R₁ may be chosen from the group consisting of:

In more specific embodiments, R₁ is:

In certain embodiments, X₁ is a halogen such as Br.

R₅ may be chosen from the group consisting of an alkylaryl having thestructure:

an aryl having the structure:

wherein m=0, 1, 2, 3, 4, 5, 6, or 7 and

where X₅ and X₆ are each independently chosen from the group consistingof hydrogen and alkyl, and

where R₇, R₈, R₉, R₁₀, and R₁₁ are each independently selected from thegroup consisting of hydrogen, halogen, alkyl, alkoxy, OH, trihalomethyl,and NO₂.

In more specific embodiments R₅ is an alkylaryl. X₁, X₂, X₃, and X₄ maybe hydrogen. Z₁ may be a lower alkyl, and the lower alkyl may be—(CH₂)_(m3)—, wherein M3=0, 1, 2, 3, or 4.

Z₃ may be a lower alkyl, and the lower alkyl may be —(CH₂)_(m4)—,wherein M4=0, 1, 2, 3, or 4. Y₁ may be O₂N or a halogen, such as Cl orBr.

In certain embodiments, R₅ is selected from the group consisting of:

R₂ may be hydrogen. Y₁ may be selected from the group consisting of O₂Nand a halogen, such as Br or Cl.

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Another aspect of the present invention concerns a method of treating acell proliferative disease comprising administering a therapeuticallyrelevant amount of a first compound of the present invention to asubject. The subject may be a mammal, and the mammal may be a human. Thefirst compound may be comprised in a pharmaceutically acceptableexcipient, diluent, or vehicle. The cell proliferative disease may becancer. The cancer may be melanoma, non-small cell lung, small celllung, lung, hepatocarcinoma, retinoblastoma, astrocytoma, glioblastoma,leukemia, blood, brain, skin, eye, tongue, gum, neuroblastoma, head,neck, breast, pancreatic, renal, bone, testicular, ovarian,mesothelioma, cervical, gastrointestinal, lymphoma, colon, or bladder.

The cell proliferative disease may be rheumatiod arthritis, inflammatorybowel disease, osteoarthritis, leiomyomas, adenomas, lipomas,hemangiomas, fibromas, vascular occlusion, restenosis, artherosclerosis,a pre-neoplastic lesion, carcinoma in situ, oral hariy leukoplakia, orpsoriasis.

In certain embodiments, stat3 activation is reduced in a cell of thesubject. c-myc expression may be reduced in a cell of the subject. Thefirst compound may be administered in combination with a therapeuticallyrelevant amount of a second compound. The second compound may be ananti-cancer compound. The first compound may be administered incombination with a surgery, a radiation therapy, or a gene therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Inhibition of MM colony formation by compounds AG490, AG1801 andAG 2019. AG1801 (12 μM) and AG 2019 (12 μM) completely inhibited MMcolony formation while AG490 (25 μM) was less effective at higherconcentrations.

FIGS. 2A-C: Inhibition of growth and survival of MM cell lines. FIG. 2A,Dose response relationships for compounds in MM-1 MM cells. The MM cellswere incubated with the indicated concentration of AG or WP compound for72 hours before cell growth and survival were estimated by MTT assay.Compounds AG1801, AG490, and WP1034 were effective in reducing thegrowth and survival of MM1 cells. FIG. 2B, Dose response relationshipsfor compounds in OCI MM cells. Compounds AG1801, AG490, and WP1034 wereeffective in reducing the growth and survival of the OCI cells. FIG. 2C,Dose response relationships for compounds in U266 MM cells. CompoundsAG1801, AG490, and WP1034 were effective in reducing the growth andsurvival of the U266 cells.

FIGS. 3A-C: Effects of AG and WP compounds on MM cell growth/survival.To determine the effect of AG and WP compounds on MM cellgrowth/survival, MM cells were incubated with the indicatedconcentration of AG or WP for 72 hours before cell growth and survivalwere estimated by MTT assay. Clear increases in the potency of AG1801,WP1034, and WP1050 can be observed compared to AG490. FIG. 3A, AG1801,WP1034, and WP1050 inhibited the growth and survival of MM1 cells anddemonstrated increased potency compared to AG490. FIG. 3B, AG1801,WP1034, and WP1050 inhibited the growth and survival of OCI cells anddemonstrated increased potency compared to AG490. FIG. 3C, AG1801,WP1034, and WP1050 inhibited the growth and survival of U266 cells anddemonstrated increased potency compared to AG490.

FIG. 4: Structures of WP1015 and WP1066 are shown.

FIG. 5: Effect of WP1066 on multiple cancer cell lines. WP1066 waseffective in reducing cell proliferation in multiple cell lines,demonstrating a potent anti-cancer effect.

FIG. 6: Structures of WP1066, WP1130, and WP1129 are shown. IC₅₀ valuesfor these compounds against MM-1 myeloma tumors are shown. This figureillustrates the improved activity of these compounds.

FIG. 7: Improved c-myc/Stat3 inhibition with WP1066, WP1130, and WP1129.The compounds were compared with regard to their Stat3/c-myc inhibitoryactivity in MM-1 cells. Strong inhibition of c-myc/Stat3 was observedfor WP1066, WP1130, and WP1129.

FIG. 8: WP1066 decreases tumor size in vivo. The results of animalstudies of human A375 melanoma tumors growing in nude mice, treated withWP1066 after tumors reached a palpable size, are shown. Animals received40 mg/kg WP1066 every other day (QM) for a total of 8 injections. Theexperiment was stopped on day 21 when the control group reached maximumtumor burden. These results indicate that WP1066 reduces tumor volume invivo.

FIG. 9: The structures of WP1119, WP1026, and WP1127 are shown. Examplesof the kinds of monosaccharides (e.g., galactose) and monosaccharidederivatives (e.g., an acetylated monosaccharide such as acetylatedgalactose, 1,2,3,4-diisopropylideno-D-galactose) that can beincorporated into the structures of compounds of the present inventionare exemplified by the structures of WP1119, WP1026, and WP1127.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Previous studies have demonstrated that cytokine pathways that activatetranscription factors (e.g., NF-kB, Stat 3) are unregulated or activatedby genetic lesions or autocrine/paracrine mechanisms in multiple tumortypes (Hallek et al., 1998; Hideshima et al., 2002). These pathwayscontribute to the tumorigenicity and progression of cancer. In thepresent invention, several compounds were synthesized, and in vitroscreening revealed that these compounds can completely block IL-6mediated Stat3 activation at low concentrations (˜1 μM). In addition,these compounds rapidly suppressed expression of the c-mycproto-oncogene, which is frequently overexpressed, rearranged or mutatedin many malignancies (Hallek et al., 1998; Selvanayagam et al., 1988;Jernberg-Wiklund et al., 1992; Kuehl et al., 1997). Structure andactivity relationships are described in the present invention fortyrphostin and tryphostin-like compounds. As compared to AG490, thesecompounds are 20 to 50-fold more active in inhibiting Jak2/Stat3signaling in IL-6 treated cells and posses rapid c-myc downregulatoryactivity. These compounds can also induce apoptosis of c-mycoverexpressing tumor cells at concentrations that parallel their c-mycdownregulatory activity. The present invention discloses compounds thatinactivate genes and signaling pathways important for tumor cellsurvival and progression, and these compounds may be used alone or incombination with other agents for the treatment of cancer.

I. Chemical Definitions

Following long-standing patent law convention, the words “a” and “an”,when used in the specification including the claims, denotes one ormore.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl offrom 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group maybe substituted or unsubstituted. When substituted, the substitutedgroup(s) is preferably hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, N(CH₃)₂,amino, or SH.

An “alkenyl” group refers to an unsaturated hydrocarbon group containingat least one carbon-carbon double bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkenyl group has 1to 12 carbons. More perferably it is a lower alkenyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkenyl group may besubstituted or unsubstituted. When substituted, the substituted group(s)is preferably hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, N(CH₃)₂, halogen,amino, or SH.

An “alkynyl” group refers to an unsaturated hydrocarbon group containingat least one carbon-carbon triple bond, including straight-chain,branched chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More perferably it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted, the substituted group(s)is preferably hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, N(CH₃)₂, amino, orSH.

An “alkoxy” group refers to an “—O-alkyl” group, where “alkyl” isdefined above.

An “aryl” group refers to an aromatic group which has at least one ringhaving a conjugated pi electron system, and includes carbocyclic aryl,heterocyclic aryl, and biaryl groups, all of which may be optionallysubstituted. Preferrably, the aryl is a substituted or unsubstitutedphenyl or pyridyl. Preferred aryl substituent(s) are halogen,trihalomethyl, hydroxyl, SH, OH, NO₂, amine, thioether, cyano, alkoxy,alkyl, and amino groups.

An “alkylaryl” group refers to an alkyl (as described above), covalentlyjoined to an aryl group (as described above). Preferably, the alkyl is alower alkyl.

“Carbocyclic aryl” groups are groups wherein the ring atoms on thearomatic ring are all carbon atoms. The carbon atoms are optionallysubstituted with preferred groups as described for aryl groups above.

“Heterocyclic aryl” groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Siutable heteroatoms include oxygen, sulfur, and nitrogen,and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo,pyrimidyl, pyrazinyl, imidazoyl, and the like, all optionallysubstituted.

An “amide” refers to a —C(O)—NH—R, where R is either alkyl, aryl,alkylaryl, or hydrogen.

A “thioamide” refers to a —C(S)—NH—R, where R is either alkyl, aryl,alkylaryl, or hydrogen.

An “ester” refers to a —C(O)—OR′, where R′ is either alkyl, aryl, oralkylaryl.

An “amine” refers to a —N(R″)R′″, where R″ and R′″ is each independentlyeither hydrogen, alkyl, aryl, or alkylaryl, provided that R″ and R′″ arenot both hydrogen.

A “thioether” refers to —S—R, where R is either alkyl, aryl, oralkylaryl.

A “sulfonyl” refers to —S(O)₂—R, where R is aryl, C(CN)═C-aryl, CH₂—CN,alkylaryl, NH-alkyl, NH-alkylaryl, or NH-aryl.

II. Tyrphostin and Tyrphostin-Like Compounds

The present invention provides tyrphostin and tyrphostin-like compoundsfor the treatment of cell proliferative diseases such as cancer.Compounds of the present invention include compounds comprising thechemical formula:

wherein R₀ is selected from the group consisting of R₁ and R₁—Z₁—; and

wherein Z₁ is alkyl; and

wherein R₁ is chosen from the group consisting of:

where X₁, X₂, X₃, and X₄, are each independently selected from the groupconsisting of hydrogen, halogen, alkyl, alkoxy, OH, trihalomethyl, andNO₂;

where Y₁ is selected from the group consisting of halogen and O₂N; and

R₂ is selected from the group consisting of alkyl, alkenyl, alkynyl,alkoxy, alkylaryl, halogen, hydrogen, OH, NO₂, thioether, amine, SH, andNH₂;

R₃ is selected from the group consisting of:

wherein Z₃ is alkyl; and

wherein m₁=1, 2, 3, or 4; and

where R₄ is chosen from the group consisting of: CN, substituted amine,CH₂S-alkyl, alkyl, and CH₂N₃;

where R₅ and R₆ are each independently chosen from the group consistingof:

monosaccharide (e.g., glucose, fructose, galactose, etc.),polysaccharide, monosaccharide derivative (e.g., an acetylatedmonosaccharide such as acetylated galactose,1,2,3,4-diisopropylideno-D-galactose) substituted and unsubstitutedaryl, and substituted and unsubstituted alkylaryl;

where Z is selected from the group consisting of NH, S, and O, and

where X₅ and X₆ are each independently chosen from the group consistingof hydrogen and lower alkyl.

In certain embodiments of the present invention, R₄ is CN. In certainembodiments, R₁ may be chosen from the group consisting of:

In more specific embodiments, R₁ is:

In certain embodiments, X₁ is a halogen such as Br.

R₅ may be chosen from the group consisting of an alkylaryl having thestructure:

an aryl having the structure:

wherein m=0, 1, 2, 3, 4, 5, 6, or 7 and

where X₅ and X₆ are each independently chosen from the group consistingof hydrogen and alkyl, and

where R₇, R₈, R₉, R₁₀, and R₁₁ are each independently selected from thegroup consisting of hydrogen, halogen, alkyl, alkoxy, OH, trihalomethyl,and NO₂.

In more specific embodiments R₅ is an alkylaryl. X₁, X₂, X₃, and X₄ maybe hydrogen. Z₁ may be a lower alkyl, and the lower alkyl may be—(CH₂)_(m3)—, wherein M3=0, 1, 2, 3, or 4.

Z₃ may be a lower alkyl, and the lower alkyl may be —(CH₂)_(m4)—,wherein M4=0, 1, 2, 3, or 4. Y₁ may be O₂N or a halogen, such as Cl orBr.

In certain embodiments, R₅ is selected from the group consisting of:

R₂ may be hydrogen. Y₁ may be selected from the group consisting of O₂Nand a halogen, such as Br or Cl.

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

Certain embodiments of the present invention relate to a compound havingthe formula:

III. Cell Proliferative Diseases

The term “cell proliferative diseases” refers to disorders resultingfrom abnormally increased and/or uncontrolled growth of cell(s) in amulticellular organism that results in harm (e.g., discomfort ordecreased life expectancy) to the multicellular organism. Cellproliferative diseases can occur in animals or humans. Cancer is anexample of a cell proliferative disease, and certain embodiments of thepresent invention are directed towards the treatment of cancer.

In certain embodiments, compounds and methods of the present inventionmay be used to treat a wide variety of cancerous states including, forexample, melanoma, non-small cell lung, small cell lung, lung,hepatocarcinoma, retinoblastoma, astrocytoma, glioblastoma, leukemia,blood, brain, skin, eye, tongue, gum, neuroblastoma, head, neck, breast,pancreatic, renal, bone, testicular, ovarian, mesothelioma, cervical,gastrointestinal, lymphoma, colon, and/or bladder. The cancer maycomprise a tumor made of cancer cells. These cancerous states mayinclude cells that are cancerous, pre-cancerous, and/or malignant.

It is also anticipated that compounds of the present invention may alsobe used to treat cell proliferative diseases other than cancer. Othercell proliferative diseases that may be treated in certain embodimentsof the present invention include, for example, rheumatiod arthritis,inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas,lipomas, hemangiomas, fibromas, vascular occlusion, restenosis,artherosclerosis, pre-neoplastic lesions (e.g., adenomatous hyperplasia,prostatic intraepithelial neoplasia), carcinoma in situ, oral hariyleukoplakia and/or psoriasis.

Additionally, compounds of the present invention may be used to treatdiseases other than hyperproliferative diseases. For example, certaintyrphostins may be useful for the treatment of hypertrophy and ischemia(U.S. Pat. No. 6,433,018) as well as hepatitis B infection (U.S. Pat.No. 6,420,338). Thus compounds of the present invention may also beuseful for the treatment of other diseases including hypertrophy,ischemia, and a viral infection (e.g., hepatitis B infection).

IV. Pharmaceutical Compositions

The anti-tumor compounds of this invention can be administered to killcertain cells involved in a cell proliferative disease, such as tumorcells, by any method that allows contact of the active ingredient withthe agent's site of action in the tumor. They can be administered by anyconventional methods available for use in conjunction withpharmaceuticals, either as individual therapeutically active ingredientsor in a combination of therapeutically active ingredients. They can beadministered alone but are generally administered with apharmaceutically acceptable carrier selected on the basis of the chosenroute of administration and standard pharmaceutical practice.

Aqueous compositions of the present invention will have an effectiveamount of the compounds to kill or slow the growth of cancer cells. Suchcompositions will generally be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium.

The terms “AG compounds” and “WP compounds” refer to specific examplesof the present invention. For example compound WP1015 is an example of aWP compound, and AG1801 is an example of an AG compound.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, orhuman, as appropriate. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredients,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients, such as other anti-cancer agents, can also beincorporated into the compositions.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g., tablets or other solids for oraladministration; time release capsules; and any other form currentlyused, including cremes, lotions, mouthwashes, inhalants, lipid carriers,liposomes and the like.

A. Parenteral Administration

The active compounds will often be formulated for parenteraladministration, e.g., formulated for injection via the intravenous,intramuscular, subcutaneous, or even intraperitoneal routes. Thepreparation of an aqueous composition that contains an anthracycline ofthe present invention as an active ingredient will be known to those ofskill in the art in light of the present disclosure. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and the preparations can also be emulsified.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

In some forms, it will be desirable to formulate the compounds in saltform, generally to improve the solubility and bioavailability and toprovide an active drug form more readily assimilated. As used herein,the term “pharmaceutically acceptable salt” refers to compounds whichare formed from acidifying a substituted anthracycline solution withsuitable physiologically tolerated acids. Suitable physiologicallytolerated acids are organic and inorganic acids, such as hydrochloricacid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalicacid, malonic acid, salicylic acid, maleic acid, methane sulfonic acid,isothionic acid, lactic acid, gluconic acid, glucuronic acid,amidosulfuric acid, benzoic acid, tartaric acid and pamoaic acid.Typically, such salt forms of the active compound will be provided ormixed prior to use.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

The active compounds may be formulated into a composition in a neutralor salt form. Pharmaceutically acceptable salts, include the acidaddition salts and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like.

The compounds of the present invention may also be formulated into acomposition comprising liposomes or any other lipid carrier. Liposomesinclude: multivesicular liposomes, multilamellar liposomes, andunilamellar liposomes.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial ad antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

In certain cases, the therapeutic formulations of the invention couldalso be prepared in forms suitable for topical administration, such asin creams and lotions. These forms may be used for treatingskin-associated diseases, such as various sarcomas.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,with even drug release capsules and the like being employable.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 mL of isotonic NaCl solutionand either added to 1000 mL of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

B. Oral Administration

In certain embodiments, active compounds may be administered orally.This is contemplated for agents which are generally resistant, or havebeen rendered resistant, to proteolysis by digestive enzymes. Suchcompounds are contemplated to include all those compounds, or drugs,that are available in tablet form from the manufacturer and derivativesand analogues thereof.

For oral administration, the active compounds may be administered, forexample, with an inert diluent or with an assimilable edible carrier, orthey may be enclosed in hard or soft shell gelatin capsule, orcompressed into tablets, or incorporated directly with the food of thediet. For oral therapeutic administration, the active compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 60% of the weight of the unit. The amount of activecompounds in such therapeutically useful compositions is such that asuitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

Upon formulation, the compounds will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms, such as those described below in specificexamples.

IV. Therapies

One of the major challenges in oncology today is the effective treatmentof a given tumor. Tumors are often resistant to traditional therapies.Thus, a great deal of effort is being directed at finding efficacioustreatment of cancer. One way of achieving this is by combining new drugswith the traditional therapies. In the context of the present invention,it is contemplated that therapies using the compounds could be used incombination with surgery, chemotherapy, radiotherapy, and/or a genetherapy.

“Effective amounts” or a “therapeutically relevant amount” are thoseamounts of a compound sufficient to produce a therapeutic benefit (e.g.,effective to reproducibly inhibit decrease, reduce, inhibit or otherwiseabrogate the growth of a cancer cell). An effective amount, in thecontext of treating a subject, is sufficient to produce a therapeuticbenefit. The term “therapeutic benefit” as used herein refers toanything that promotes or enhances the well-being of the subject withrespect to the medical treatment of the subject's cell proliferativedisease. A list of nonexhaustive examples of this includes extension ofthe patients life by any period of time; decrease or delay in theneoplastic development of the disease; decrease in hyperproliferation;reduction in tumor growth; delay of metastases; reduction in theproliferation rate of a cancer cell, tumor cell, or any otherhyperproliferative cell; induction of apoptosis in any treated cell orin any cell affected by a treated cell; and/or a decrease in pain to thesubject that can be attributed to the patient's condition.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 General Method for Synthesis of Compounds

N-(Phenylalkyl)cinnamides were prepared by the following generalprocedure. Benzylamine (3.0 g, 28 mmol) and ethyl cyanoacetate (4.7 g,42 mmol) in acetonitrile (20 mL) was stirred and reflux for 4 hr.Benzylamine in this general procedure can be replaced by any othersubstituents depicted as R₃ above. The solvent was removed in vacuo togive an oil which solidified upon standing. Precipitation (EtOAc)resulted in 3.28 g (68%) of an off-white powder corresponding toN-benzylcyanoacetamide as an intermediate. A mixture ofN-benzylcyanomethylamide (1.3, 7.5 mmol), 3,4-dihydroxybenzaldehyde (1.1g, 8.2 mmol), and piperidine (catalytic, 5 drops) was stirred at refluxfor 3 hr. Flash chromatography (EtOAc) followed by two recrystalizations(H₂O/EtOH) yielded product as a white powder 0.8 g (36%).

Example 2 Synthesis of Compounds

Using the protocol detailed in Example 1, the following compounds weresynthesized, and data regarding the synthesis of these compounds arepresented below.

AG1801 was synthesized. ¹H-NMR (CDCl₃, 500 MHz, δ): 8.49 (s, 1H, H-3),7.39 (d, 2H, J=8.7 Hz, H-3′,5′), 8.12 (d, 2H, J=7.5 Hz, H-2′,6′),7.43-7.39 (5H, H aromat. from benzyl), 6.75 (bs, 1H, NH), 4.68 (d, 2H,J=5.7 Hz, CH₂). Elemental. Anal. Calcd. For C₁₇H₁₃N₃O₃, Theor. C, 66.44;H, 4.26; N, 13.67. Found: C, 65.70; H, 4.27; N, 13.45. m.p. 165-166° C.

WP1002 was synthesized. ¹H-NMR (CDCl₃, 500 MHz, δ): 8.23 (s, 1H, H-3),7.83 (d, 2H, J=8.6 Hz, H-2′,6′), 7.39-7.28 (5H, H aromat. from benzyl),6.69 (d, 2H, J=8.6 Hz, H-3′,5′), 6.58 (bs, 1H, NH), 4.62 (d, 2H, J=5.7Hz, CH₂). 4.30 (bs, 2H, NH₂)

WP1003 was synthesized. ¹H-NMR (CDCl₃, 500 MHz, δ): 8.30 (s, 1H, H-3),7.92 (d, 2H, J=8.7 Hz, H-2′,6′), 7.71 (s, 1H, NH), 7.66 (d, 2H, J=8.4Hz, H-3′,5′), 7.42-7.28 (5H, H aromat. from benzyl), 6.70 (bs, 1H, NH),4.62 (d, 2H, J=5.7 Hz, CH₂). 2.22 (s, 3H, CH₃)

WP1004 was synthesized. ¹H-NMR (CDCl₃, 500 MHz, δ): 8.30 (s, 1H, H-3),7.94 (d, 2H, J=8.7 Hz, H-2′,6′), 7.68 (d, 2H, J=8.6 Hz, H-3′,5′), 7.51(s, 1H, NH), 7.43-7.39 (5H, H aromat. from benzyl), 6.67 (t, 1H, J=5.3Hz, NH), 4.62 (d, 2H, J=5.7 Hz, CH₂). 2.41 (t, 2H, J=7.4 Hz, CH₂), 1.73(m, 2H, CH₂), 1.42 (m, 2H, CH₂), 0.96 (t, 3H, J=7.4 Hz, CH₃)

WP1005 was synthesized. ¹H-NMR (CDCl₃, 500 MHz, δ): 8.31 (s, 1H, H-3),7.94 (d, 2H, J=8.7 Hz, H-2′,6′), 7.68 (d, 2H, J=8.6 Hz, H-3′,5′), 7.41(s, 1H, NH), 7.40-7.28 (5H, H aromat. from benzyl), 6.66 (t, 1H, J=5.7Hz, NH), 5.35 (m, 2H, CH═CH), 4.62 (d, 1H, J=5.7 Hz, CH₂). 2.40 (t, 2H,J=7.4 Hz, CH₂), 1.73 (m, 4H, CH₂), 1.75 (m, 2H, CH₂), 1.31 (m, 22H,CH₂), 0.89 (t, 3H, J=7.0 Hz, CH₃)

WP1009 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 11.88 (bs, 1H,H-1′), 8.64 (t, 1H, J=5.8 Hz, NH), 8.07 (s, 1H, H-3), 7.36-7.22 (m, 7H,H-3′,5′ and Haromat. from benzyl), 6.41 (bs, 1H, H-4′), 4.39 (d, 2H,J=6.0 Hz, CH₂). m.p. 216-217° C.

WP1010 was synthesized. ¹H-NMR (DMSO-d₆, 500 MHz, δ): 10.42 (bs, 1H,NHSO₂Me), 8.94 (bs, 1H, NH), 8.15 (s, 1H, H-3), 7.98 (d, 2H, J=8.5 Hz,H-2′,6′), 7.43-7.39 (7H, H-3′,5′ and H aromat. from benzyl), 4.43 (d,1H, J=5.4 Hz, CH₂), 3.15 (s, 3H. Me).

WP1006 was synthesized. ¹H-NMR (CDCl₃, 500 MHz, δ): 8.35 (s, 1H, H-3),7.98 (ddd, 2H, J=8.6 Hz, J=5.4 Hz, J=5.1 Hz, H-2′,6′), 7.40-7.31 (5H, Haromat. from benzyl), 7.20 (dd, 2H, J=8.5 Hz, J=11.5 Hz, H-3′,5′), 6.71(bs, 1H, NH), 4.62 (d, 2H, J=5.8 Hz, CH₂). Elemental. Anal. Calcd. forC₁₇H₁₃FN₂O, Theor. C, 72.85; H, 4.67; F, 6.78; N, 9.99. Found: C, 72.86;H, 4.65; F, 6.68; N, 9.80. m.p. 150-151° C.

WP1007 was synthesized. ¹H-NMR (CDCl₃, 500 MHz, δ): 8.33 (s, 1H, H-3),8.00 (d, 2H, J=8.6 Hz, H-3′,5′), 7.40-7.27 (5H, H aromat. from benzyl),7.14 (dd, 2H, J=7.0 Hz, J=1.9 Hz, H-2′,6′), 6.66 (bs, 1H, NH), 4.63 (d,1H, J=5.8 Hz, CH₂). m.p. 147-149° C.

WP1011 was synthesized. ¹H-NMR (CDCl₃, 300 MHz, δ): 8.31 (s, 1H, H-3),7.98 (d, 2H, J=8.6 Hz, H-3′,5′), 7.63 (d, 2H, J=8.5 Hz, H-2′,6′),7.40-7.26 (5H, H aromat. from benzyl), 6.67 (bs, 1H, NH), 4.61 (d, 2H,J=5.8 Hz, CH₂). m.p. 177-178° C.

WP1012 was synthesized. ¹H-NMR (DMSO-d₆, 300 MHz, δ): 11.58 (bs, 1H,H-1′), 8.86 (t, 1H, J=5.7 Hz, NH), 8.28 (s, 1H, H-3), 8.25 (bs, 1H,H-4′), 7.83 (dd, 1H, J=8.6 Hz, J=1.7 Hz, H-8′), 7.57 (d, 1H, J=8.7 Hz,H-7′), 7.50 (dd, 1H, J=2.8 Hz, H-2′), 7.35-7.21 (m, 5H, H aromat. frombenzyl), 6.60 (bs, 1H, H-3′), 4.44 (d, 2H, J=5.7 Hz, CH₂). m.p. 199-200°C.

WP1013 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 10.93 (s, 1H, OH),8.66 (d, 1H, J=2.3 Hz, H-6), 8.31 (s, 1H, H-7), 8.26 (dd, 1H, J=8.9 Hz,J=2.2 Hz, H-2), 7.40-7.29 (6H, H-3 and H aromat. from benzyl), 6.66 (bs,1H, NH), 4.61 (d, 1H, J=5.7 Hz, CH₂). m.p. 158-159° C.

WP1014 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 10.56 (s, 1H, OH),8.33 (s, 1H, H-3), 8.23 (d, 1H, J=8.8 Hz, H-3′), 7.63 (d, 2H, J=2.0 Hz,H-6′), 7.51 (dd, 1H, J=8.8 Hz, J=2.0 Hz, H-2′), 7.40-7.31 (5H, H aromat.from benzyl), 6.71 (bs, 1H, NH), 4.62 (d, 2H, J=6.0 Hz, CH₂). Elemental.Anal. Calcd. For C₁₇H₁₃N₃O₄, Theor. C, 63.16; H, 4.05; N, 13.00. Found:C, 62.88; H, 4.15; N, 12.80. m.p. 171-172° C.

WP1015 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 8.26 (s, 1H, H-3),7.67 (dd, 1H, J=7.6 Hz, H-5′), 7.60 (dd, 1H, J=7.4 Hz, J=1 Hz, H-4′),7.58 (dd, 1H, J=7.7 Hz, J=1 Hz, H-6′), 7.40-7.26 (m, 5H, H, H aromat.from benzyl), 6.91 (bs, 1H, NH), 4.62 (d, 2H, J=5.8 Hz, CH₂). m.p.182-183° C.

WP1016 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 8.50 (s, 1H, H-3),8.43 (dd, 1H, J=6.8 Hz, J=2.2 Hz, H-6′), 7.81 (ddd, 1H, J=8.7 Hz, J=5.2Hz, J=2.0 Hz, H-3′), 7.40-7.31 (m, 5H, H arom. from benzyl), 6.96 (dd,1H, J=9.7 Hz, J=8.7 Hz, H-4′), 6.68 (bs, 1H, NH), 4.62 (d, 2H, J=5.7 Hz,CH₂). Elemental. Anal. Calcd. For C₁₇H₁₂FIN₂O, Theor. C, 50.27; H, 2.98;F, 4.68; I, 31.24; N, 6.90. Found: C, 50.68; H, 3.22; N, 6.73; F, 4.46;I, 30.33. m.p. 138-139° C.

WP1017 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 8.50 (s, 1H, H-3),8.43 (dd, 1H, J=6.8 Hz, J=2.2 Hz, H-6′), 7.81 (ddd, 1H, J=8.7 Hz, J=5.2Hz, J=2.0 Hz, H-3′), 7.40-7.31 (m, 5H, H arom. from benzyl), 6.96 (dd,1H, J=9.7 Hz, J=8.7 Hz, H-4′), 6.68 (bs, 1H, NH), 4.62 (d, 2H, J=5.7 Hz,CH₂). m.p. 183-184° C.

WP1018 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 8.38 (s, 1H, H-3),8.06 (d, 2H, J=8.6 Hz, H-2′,6′), 7.93 (s, 1H, H-2″), 7.53 (d, 2H, J=8.6Hz, H-3′,5′), 7.40-7.30 (6H, H-5″ and H aromat. from benzyl), 7.25 (d,1H, J=4.1 Hz, H-4″), 6.77 (d, 1H, J=6.2 Hz, NH), 4.62 (d, 2H, J=5.8 Hz,CH₂). m.p. 187-188° C.

WP1019 was synthesized. ¹H-NMR (DMSO-d₆ 400 MHz, δ): 9.04 (t, 1H, J=6.0Hz, NH), 8.29 (bs, 2H, OH), 8.22 (s, 1H, H-3), 7.94 (d, 2H, J=8.5 Hz,H-2′,6′), 7.90 (d, 2H, J=8.4 Hz, H-3′,4′), 7.37-7.24 (m, 5H, H aromat.from benzyl), 4.43 (d, 2H, J=5.8 Hz, CH₂)

WP1020 was synthesized. ¹H-NMR (DMSO-d₆ 300 MHz, δ): 12.87 (bs, 1H, NH),12.45 (bs, 1H, NH), 7.82 (s, 1H, H-3), 7.49 (s, 1H, H-3′), 7.26-7.11 (m,6H, H-5′ and H arom from benzyl), 4.37 (d, 2H, J=5.5 Hz, CH₂). m.p.230-231° C.

WP1021 was synthesized. ¹H-NMR (DMSO-d₆, 400 MHz, δ): 12.42 (bs, 1H,H-1′), 8.83 (dd, 1H, J=5.7 Hz, NH), 8.49, 8.47 (2s, 1H ea, H-2′,3), 8.01(d, 1H, J=2.0 Hz, H-4′), 7.56 (d, 1H, J=8.5 Hz, H-7′), 7.33-7.32 (5H, Haromat. from benzyl), 7.26 (dd, 1H, J=8.6 Hz, J=2.1 Hz, H-6′), 4.44 (d,2H, J=6.0 Hz, CH₂). Elemental. Anal. Calcd. For C₁₉H₁₄ClN₃O, Theor. C,67.96; H, 4.20; Cl, 10.56; N, 12.51. Found: C, 68.15; H, 4.34; Cl,10.78; N, 12.31. m.p. 229-230° C.

WP1022 was synthesized. ¹H-NMR (DMSO-d₆, 300 MHz, δ): 12.46 (bs, 1H,H′), 8.87 (dd, 1H, J=5.9 Hz, NH), 8.50 (s, 1H, H-3), 8.49 (d, 1H, J=2.6Hz, H-2′), 8.17 (d, 1H, J=1.7 Hz, H-4′), 7.53 (d, 1H, J=8.6 Hz, H-7′),7.40 (dd, 1H, J=8.5 Hz, J=2 Hz, H-6′), 7.36-7.23 (5H, H aromat. frombenzyl), 4.43 (d, 2H, J=5.9 Hz, CH₂). m.p. 224-225° C.

WP1026 was synthesized. ¹H-NMR (CDCl₃, 300 MHz, δ): 8.30 (s, 1H, H-3),8.01 (s, 1H, H-6′), 7.89 (d, 1H, J=8.0 Hz, H-4′), 7.66 (d, 1H, J=8.1 Hz,H-2′), 7.40-7.30 (m, 6H, H-3′ and H aromat. from benzyl), 6.68 (bs, 1H,NH), 4.61 (d, 2H, J=5.7 Hz, CH₂). m.p. 150-151° C.

WP1027 was synthesized. ¹H-NMR (DMSO-d₆ 300 MHz, δ): 8.81 (bs, 1H, OH),8.74 (t, 1H, J=5.8 Hz, NH), 7.32-7.09 (m, 5H, H aromat. from benzyl),6.66 (d, 1H, J=2.3 Hz, H-2′), 6.64 (d, 1H, J=8.3 Hz, H-5′), 6.50 (dd,1H, J=8.1 Hz, J=2.1 Hz, H-6′), 4.32 (dd, 1H, J=15.2 Hz, J=6.3 Hz, CH₂),4.22 (dd, 1H, J=15.2 Hz, J=5.5 Hz, CH₂), 3.87 (dd, 1H, J=7.1 Hz, CH),2.98 (dd, 1H, J=13.4 Hz, J=7.3 Hz, 3-CH₂), 2.90 (dd, 1H, J=13.4 Hz,J=8.2 Hz, 3-CH₂). m.p. 131-132° C.

WP1034 was synthesized. ¹H-NMR (DMSO-d₆, 400 MHz, δ): 8.41 (s, 1H, H-3),8.33 (d, 2H, J=8.8 Hz, H-3′,5′), 8.05 (d, 2H, J=8.7 Hz, H-2′,6′),7.41-7.26 (m, 5H, H-aromat. from benzyl), 6.60 (d, 1H, J=7.6 Hz, NH),5.28-5.21 (m, 1H, CH), 1.62 (d, 3H, J=6.9 Hz, CH₃). m.p. 172-173° C.

WP1035 was synthesized. ¹H-NMR (DMSO-d₆, 300 MHz, δ): 11.07 (s, 1H, OH),8.90 (t, 1H, J=5.8 Hz, NH), 8.10 (s, 1H, H-3), 7.85 (dd, 1H, J=12.5 Hz,J=2.1 Hz, H-6′), 7.69 (dd, 1H, J=8.5 Hz, J=2.0 Hz, H-5′), 7.37-7.22 (m,5H, H-arom. from benzyl), 7.12 (d, 1H, J=8.8 Hz, H-2′), 4.41 (d, 2H,J=5.9 Hz, CH₂). m.p. 211-212° C.

WP1036 was synthesized. ¹H-NMR (DMSO-d₆, 400 MHz, δ): 10.71 (bs, 1H,OH), 8.86 (t, 1H, J=6.2 Hz, NH), 8.05 (s, 1H, H-3), 7.96 (d, 1H, J=1.9Hz, H-2′), 7.66 (d, 1H, J=2.0 Hz, H-6′), 7.34-7.21 (m, 5H, H arom. frombenzyl), 4.40 (d, 2H, J=5.8 Hz, CH₂), 3.85 (s, 3H, OMe). m.p. 206-207°C.

WP1037 was synthesized. ¹H-NMR (CDCl₃, 500 MHz, δ): 7.37-7.21 (m, 5H, Haromat. from benzyl), 7.11 (d, 1H, J=11.4 Hz, H-3) 6.40 (bs, 1H, NH),4.56 (d, 1H, J=5.8 Hz, CH₂), 2.10-2.03 (m, 1H, H-1′), 1.30 (ddd, 2H,J=12.8 Hz, J=7.6 Hz, J=5.0 Hz, CH₂), 0.98 (ddd, 2H, J=8.9 Hz, J=7.3 Hz,J=4.6 Hz, CH₂). m.p. 106-107° C.

WP1038 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 10.54 (s, 1H, OH),8.27 (s, 1H, H-3), 8.21 (d, 1H, J=8.8 Hz, H-5′), 7.60 (d, 1H, J=2.2 Hz,H-2′), 7.50 (dd, 1H, J=8.9 Hz, J=2.0 Hz, H-6′), 7.40-7.20 (5H, H aromat.from benzyl), 6.60 (d, 1H, J=6.5 Hz, NH), 5.28-5.21 (m, 1H, CH), 1.60(d, 1H, J=6.9 Hz, CH₃). m.p. 178-179° C.

WP1040 was synthesized. ¹H-NMR (DMSO-d₆, 400 MHz, δ): 10.07, 9.54 (2s,1H ea, OH), 8.62 (d, 1H, J=7.8 Hz, NH), 7.92 (s, 1H, H-3), 7.52 (d, 1H,J=2.0 Hz, H-2′), 7.37-7.20 (5H, H aromat. from benzyl), 7.26 (dd, 1H,J=8.3 Hz, J=2.1 Hz, H-6′), 6.86 (d, 1H, J=8.3 Hz, H-5′), 5.06-4.99 (m,1H, CH), 1.45 (d, 1H, J=7 Hz, CH₃). m.p. 141-142° C.

WP1041 was synthesized. ¹H-NMR (CDCl₃, 500 MHz, δ): 8.20 (s, 1H, H-3),7.98 (d, 2H, J=9 Hz, H-2′,6′), 7.37-7.28 (m, 5H, H-arom. from benzyl),6.69 (d, 2H, J=9 Hz, H-3′,5′), 6.54 (bs, 1H, NH), 4.59 (d, 2H, J=5.7 Hz,CH₂), 3.09 (s, 6H, CH₃N). m.p. 185-186° C.

WP1042 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 8.27 (s, 1H, H-3),7.69 (d, 1H, J=2.1 Hz, H-6′), 7.44-7.26 (12H, H-2′, 5′ and H aromat.from benzyls), 6.95 (d, 1H, J=8.4 Hz, H-3′), 6.6 (t, 1H, J=5.7 Hz, NH),5.24 (s, 2H, H-7′), 4.61 (d, 2H, J=5.7 Hz, CH₂), 3.94 (s, 3H, OMe). m.p.132-133° C.

WP1043 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 8.72 (d, 1H. J=1.7Hz, H-4′), 8.55 (s, 1H, H-3), 8.15 (m, 2H, H-5, H-2′), 7.54 (ddd, 1H,J=8.2 Hz, J=1.3 Hz, H-6′), 7.46 (m, 2H, H-1′,8′), 7.38-7.29 (m, 6H, H-7′and H aromat. from benzyl), 6.66 (t, 1H, J=5.0 Hz, NH), 4.65 (d, 2H,J=5.7 Hz, CH₂), 4.40 (q, 2H, J=7.3 Hz, H-10′), 1.47 (t, 3H, H-11′). m.p.182-183° C.

WP1044 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 8.25 (s, 1H, H-3),8.00 (s, 1H, H-2′), 7.54 (d, 1H, J=2 Hz, H-5′), 7.39-7.29 (5H, H aromat.from benzyl), 7.18 (d, 1H, J=2 Hz, H-4′), 6.56 (bs, 1H, NH), 4.59 (d,2H, J=5.7 Hz, CH₂). m.p. 188-189° C.

WP1049 was synthesized. ¹H-NMR (DMSO-d₆, 500 MHz, δ): 8.75 (t, 1H, J=5.8Hz, NH), 8.26 (dd, 2H, J=8.8 Hz, J=1.9 Hz, H-3′,5′), 7.84 (dd, 2H, J=8.7Hz, J=2.4 Hz, H-2′,6′), 7.59 (d, 1H, J=15.9 Hz, H-3), 7.38-7.24 (m, 5H,H arom from benzyl), 6.89 (d, 1H, J=15.9 Hz, H-2), 4.43 (d, 2H, J=5.9Hz, CH₂). m.p. 193-194° C.

WP1050 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 8.38 (s, 1H, H-3),8.33 (d, 2H, J=8.9 Hz, H-3′,5′), 8.04 (d, 2H, J=8.9 Hz, H-2′,6′),7.41-7.29 (5H, H aromat. from benzyl), 6.60 (d, 1H, J=8.2 Hz, NH),5.28-5.21 (m, 1H, CH), 1.62 (d, 3H, J=7.0 Hz, CH₃). m.p. 173-174° C.

WP1051 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 10.56 (bs, 1H, OH),8.28 (s, 1H, H-3), 8.21 (d, 1H, J=8.8 Hz, H-5′), 7.61 (d, 1H, J=1.8 Hz,H-2′), 7.50 (dd, 1H, J=8.8 Hz, J=1.8 Hz, H-6′), 7.41-7.30 (5H, H aromat.from benzyl), 6.62 (d, 1H, J=8.0 Hz, NH), 5.27-5.21 (m, 1H, CH), 1.61(d, 3H, J=6.9 Hz, CH₃). m.p. 176-177° C.

WP1052 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 10.93 (s, 1H, OH),8.64 (d, 1H, J=2.3 Hz, H-2′), 8.25 (s, 1H, H-3), 8.24 (dd, 1H, J=8.9 Hz,J=2.3 Hz, H-6′), 7.40-7.28 (6H, H-5′ and H aromat. from benzyl), 6.54(d, 1H, J=6.4 Hz, NH), 5.28-5.21 (m, 1H, CH), 1.61 (d, 3H, J=6.9 Hz,CH₃). m.p. 182-183° C.

WP1053 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 8.34 (s, 1H, H-3),8.57 (dd, 1H, J=2.1 Hz, H-6′), 7.50 (d, 1H, J=7.8 Hz, H-2′), 7.40-7.30(10H, H aromat. from benzyls), 7.16 (dd, 1H, J=7.5 Hz, J=2.4 Hz, H-3′),6.67 (bs, 1H, NH), 5.12 (s, 2H, H-7′), 4.62 (d, 2H, J=5.7 Hz, CH₂). m.p.190-191° C.

WP1054 was synthesized. ¹H-NMR (DMSO-d₆, 500 MHz, δ): 11.4 (s, 1H, OH),8.89 (t, 1H, J=5.8 Hz, NH), 8.09 (s, 1H, H-3), 8.05 (d, 1H, J=2.2 Hz,H-2′), 7.83 (dd, 1H, J=8.7 Hz, J=2.2 Hz, H-6′), 7.40-7.22 (5H, H aromat.from benzyl), 7.12 (d, 1H, J=8.6 Hz, H-5′), 4.41 (d, 1H, J=5.9 Hz, CH₂).m.p. 213-214° C.

WP1055 was synthesized. ¹H-NMR (DMSO-d₆, 300 MHz, δ): 9.06 (t, 1H, J=5.8Hz, NH), 8.77 (d, 1H, J=4.6 Hz, H-3′), 8.18 (s, 1H, H-3), 7.99 (ddd, 1H,J=7.8 Hz, J=2.2 Hz, H-5′), 7.85 (d, 1H, J=7.7 Hz, H-6′), 7.55 (dd, J=7.5Hz, J=4.9 Hz. H-4′), 7.38-7.23 (5H, H aromat. from benzyl), 4.44 (d, 1H,J=5.9 Hz, CH₂). m.p. 184-185° C.

WP1060 was synthesized. ¹H-NMR (CDCl₃, 300 MHz, δ): 8.13 (dd, 2H, J=8.7Hz, J=2.4 Hz, H-3′,5′), 7.42 (d, 2H, J=8.7 Hz, H-2′,6′), 7.37-7.17 (m,5H, H arom from benzyl), 6.78 (bs, 1H, NH), 4.69 (dd, 1H, J=7.1 Hz,J=4.5 Hz, H-2), 4.49 (dd, 1H, J=14.7 Hz, J=6.1 Hz, CH₂), 4.39 (dd, 1H,J=14.7 Hz, J=5.7 Hz, CH₂), 3.55 (dd, 1H, J=14.2 Hz, J=4.5 Hz H-3), 3.46(dd, 1H, J=14.2 Hz, J=7.1 Hz H-3). m.p. 129-130° C.

WP1063 and WP1064 were synthesized. ¹H-NMR (DMSO-d₆, 300 MHz, δ): 8.23(t, 1H, J=6.1 Hz, NH), 8.16 (d, 2H, J=8.8 Hz, H-3′,5′), 7.64 (d, 2H,J=8.6 Hz, H-2′,6′), 7.29-7.18 (m, 5H, Harom from benzyl), 5.68 (d, 1H,J=6.2 Hz, OH), 5.49 (d, 1H, J=6.9 Hz, OH), 5.07 (dd, 1H, J=6.1 Hz, J=2.9Hz, CH), 4.31 (d, 2H, J=3.9 Hz, CH₂), 4.09 (dd, 1H, J=7.0 Hz, J=2.9 Hz,CH). m.p. 160-161° C.

WP1065 was synthesized. ¹H-NMR (CDCl₃, 300 MHz, δ): 8.81 (d, 1H, J=4.3Hz, H-3′), 8.30 (s, 1H, H-3), 7.80 (ddd, 1H, J=7.7 Hz, J=0.8 Hz, H-5′),7.61 (d, 1H, J=7.7 Hz, H-6′), 7.42-7.28 (m, 6H, H-4′ and H aromat. frombenzyl), 6.79 (d, 1H, J=6.8 Hz, NH), 5.30-5.21 (m, 1H, CH), 1.61 (d, 3H,J=6.9 Hz, CH₃). m.p. 153-154° C.

WP1066 was synthesized. ¹H-NMR (CDCl₃, 300 MHz, δ): 8.20 (s, 1H, H-3),7.66 (dd, 1H, J=7.6 Hz, H-5′), 7.59-7.56 (m, 2H, H4′,6′), 7.37-7.26 (m,5H, H aromat. From benzyl), 6.80 (d, 1H, J=7.0 Hz, NH), 5.29-5.20 (m,1H, CH), 1.61 (d, 3H, J=6.9 Hz, CH₃). m.p. 143-144° C.

WP1067 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 8.80 (s, 1H, H-3),8.27 (dd, 1H, J=7.8 Hz, J=0.6 Hz, H-3′), 7.80-7.76 (m, 2H, H-5′,6′),7.72-7.67 (m, 1H, H-4′), 7.41-7.26 (m, 5H, H aromat. from benzyl), 6.69(bs, 1H, NH), 4.62 (d, 1H, J=5.7 Hz, CH₂).

WP1069 was synthesized. ¹H-NMR (CDCl₃, 300 MHz, δ): 9.09 (d, 1H, J=2.2Hz, H-3′), 8.80 (s, 1H, H-3), 8.62 (dd, 1H, J=8.5 Hz, J=2.3 Hz, H-5′),7.97 (d, 1H, J=8.5 Hz, H-6′), 7.42-7.31 (m, 6H, H aromat. from benzyl),6.69 (bs, 1H, NH), 4.63 (d, 2H, CH₂).

WP1076 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 8.59 (s, 1H, H-3),8.01 (d, 1H, J=9.0 Hz, H-6′), 7.39-7.31 (m, 5H, H aromat. from benzyl),4.77 (d, 1H, J=2.7 Hz, H-3′), 6.89 (dd, 1H, J=8.9 Hz, J=2.7 Hz, H-5′),6.58 (bs, 1H, NH), 4.60 (d, 2H, J=5.7 Hz, CH₂), 3.13 (s, 6H, 2CH₃).

WP1074 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 8.71 (dd, 1H, J=1.9Hz, H-2′), 8.44 (s, 1H, H-3), 8.38 (ddd, 1H, J=8.2 Hz, J=2.1 Hz, J=0.8Hz, H-4′), 8.27 (d, 1H, J=7.8 Hz, H-6′), 7.72 (dd, J=8.0 Hz, H-5′),7.40-7.30 (m, 5H, H aromat. from benzyl), 6.71 (bs, 1H, NH), 4.63 (d,1H, J=5.8 Hz, CH₂).

WP1073 was synthesized. ¹H-NMR (DMSO-d₆, 400 MHz, δ): 11.44 (bs, 1H,OH), 9.07 (dd, 1H, J=5.9 Hz, NH), 8.59 (s, 1H, H-3), 8.22 (d, 1H, J=9.0Hz, H-3′), 7.38-7.25 (m, 5H, H aromat. from benzyl), 7.12 (d, 1H, J=2.6Hz, H-6′), 7.08 (dd, J=9.1 Hz, J=2.6 Hz, H-4′), 4.44 (d, 2H, J=5.9 Hz,CH₂).

WP1077 was synthesized. ¹H-NMR (CDCl₃, 400 MHz, δ): 8.35 (d, 1H, J=2 Hz,H-2′), 8.34 (s, 1H, H-3), 8.09 (dd, 1H, J=8.4 Hz, J=2.4 Hz, H-6′), 7.70(d, 1H, J=8.4 Hz, H-5′), 7.40-7.26 (m, 5H, H aromat. from benzyl), 6.68(bs, 1H, NH), 4.62 (d, 2H, J=5.6 Hz, CH₂).

WP1075 was synthesized. ¹H-NMR (DMSO-d₆, 400 MHz, δ): 9.08 (dd, 1H,J=6.1 Hz, NH), 8.98 (d, 1H, J=2.3 Hz, H-2′), 4.96 (dd, 1H, J=4.8 Hz,J=1.6 Hz, H-4′), 8.37 (ddd, 1H, J=8.2 Hz, J=2.0 Hz, H-6′), 8.26 (s, 1H,H-3), 7.60 (dd, 1H, J=8.2 Hz, J=4.8 Hz, H-5′), 7.35-7.23 (m, 5H, Haromat. from benzyl), 4.43 (d, 2H, J=6.0 Hz, CH₂).

WP1119 was synthesized. ¹H-NMR (CDCl₃, 300 MHz, δ): 8.20 (s, 1H, H-3),7.70-7.62 (m, 2H, H-4′, H-6′), 7.59-7.56 (m, 1H, H-5′), 7.14 (m, 1H,NH), 5.54 (d, 1H, H-1″, J=4.97 Hz), 4.63 (dd, 1H, H-3″, J=2.23, J=7.94Hz), 4.32 (dd, 1H, H-2″, J=2.23, J=4.97 Hz), 4.29-4.26 (m 1H, H-4″),4.03-4.00 (m, 1H, H-5″), 3.93-3.85 (m, 1H, H-6″), 3.54-3.45 (m, 1H,H-6″), 1.51 (s, 3H CH₃″), 1.49 (s, 3H, CH₃″), 1.36 (s, 3H CH₃″), 1.32(s, 3H, CH₃″).

WP1126 was synthesized. ¹H-NMR (DMSO, 300 MHz, δ): 8.42 (m, 1H, NH),8.10 (s, 1H, H-3), 7.97-7.88 (m, 2H, H-4′, H-6′), 7.82-7.79 (m, 1H,H-5′), 5.75-6.50 (bs, 1H, OH), 4.94 (d, 1H, H-1″, J=2.66 Hz), 4.49-3.77(m, 3H, OH), 4.02-3.98 (m, 1H), 3.67 (m, 1H), 3.60-3.50 (m 2H),3.40-3.36 (m, 2H, H-6″).

WP1127 was synthesized. ¹H-NMR (CDCl₃, 300 MHz, δ): 8.21 (s, 1H, H-3),7.72-7.59 (m, 3H, H-4′, H-5′, H-6′), 6.95-6.91 (m, 1H, NH), 6.41 (m, 1H,H-1″), 5.48 (m, 1H), 5.37 (m, 2H), 4.34-4.29 (m, 1H, H-5″), 3.66-3.47(m, 2H, H-6″), 2.24 (s, 3H, CH₃), 2.17 (s, 3H, CH₃), 2.04 (s, 3H, CH₃),2.03 (s, 3H, CH₃).

Example 3 Compounds Display Potent Anti-Cancer Effects

IL-6 stimulates Stat3 phosphorylation in multiple myeloma (MM) andnon-Hodgkin's lymphoma (NHL) cells. In FIG. 1, MM cells (MM-1, 8226,8226/S, U266) or NHL (DBr, DB, DS, LP, LR, Mino, MS, FN, Jeko, JM) cellswere treated with IL-6 (10 ng/ml) for 10 min before cell lysates wereprepared and evaluated for Stat3 tyrosine phosphorylation by immunoblot(anti-pY705-Stat3 from Cell Signaling). IL-6 stimulated Stat3phosphorylation in all MM cell lines and in 5 out of 10 NHL cell lines(specifically, in DB, DS, LP, FN, and JM cells). It should be noted thatU266 cells expressed constitutively activated Stat3 that was furtherstimulated by exogenous addition of IL-6.

To examine the effect of newly synthesized AG and WP compounds oncytokine-mediated Stat activation, multiple myeloma (MM-1) cells werepretreated with AG490 or AG1801 at 2.5, 12, and 25 μM concentrations(for 2 hr) before stimulating cells with IL-6 or IFN-α for 10 min. Stat3and Stat1 expression and activation were examined by immunoblotting.AG1801 (at 12 and 25 μM) was effective in suppressing IL-6 signalingwithout effecting IFN-α, signaling. AG2019 had similar activity at 12and 25 μM. AG490 was inactive under these conditions.

To determine whether AG1801 effects caspase activation in MM cells,OCI-My5 cells were treated as described above before cell lysates wereexamined for casapase activation and PARP cleavage. At 12.5 and 25 μM,AG1801 activated both upstream and downstream caspases (i.e., caspase 3and caspase 8), and AG1801 increased PARP clevage. At 12.5 and 25 μM,AG490 was ineffective in caspase activation and PARP clevage in thesecells.

To determine whether these compounds effect primary MM colony growthbone marrow aspirates from MM patients were partially-purified bymagnetic bead separation and analyzed for immunoglobulin heavy-chaingene rearrangement by PCR. Cells were grown as colonies inmethyl-cellulose in the presence or absence of AG compound as noted for7 to 10 day s. Control colonies were examined for Ig heavy chain generearrangement to confirm the clonal nature of the population. As shownin FIG. 1, AG1801 and AG 2019 completely inhibited MM colony formationwhile AG490 was less effective at higher concentrations.

To determine the effect of compounds on both Stat3 activation and c-mycprotein expression MM-1, OCI-My5 and U266 cells were incubated with 25μM of AG1801, AG490, WP1038, WP1039, WP1051, or WP1052 for 2 hoursbefore cells were stimulated with 2 ng/ml IL-6 for 10 min. Cell lysateswere prepared and immunoblotted for p-Stat3, Stat3, c-myc and actin (asa control). Both Stat3 activation and c-myc expression were effected byWP and AG compounds. IL-6 stimulated Stat3 activation but did notsignificantly c-myc expression.

To determine the effect of AG and WP compounds on MM cellgrowth/survival, MM cells were incubated with the indicatedconcentration of AG or WP for 72 hours before cell growth and survivalwere estimated by MTT assay. As shown in FIGS. 2A-C, compounds active indownregulating c-myc and blocking IL-6 mediated Stat3 activation wereeffective in reducing the growth and survival of MM cell lines.

To determine the temporal effects and mechanism of action of AG490 andAG1801 action on c-myc expression in MM cells, MM-1 cells were incubatedwith 0, 25, or 50 μM of AG490 or AG1801 and harvested at 30, 60, or 120min. Lysates were immunoblotted for c-myc or actin as a protein loadingcontrol. AG1801 rapidly reduced c-myc expression in MM-1 cells at 25 and50 μM at all measured incubation durations. In contrast, AG490 wascompletely unable to affect c-myc expression at the concentrations andincubation durations tested. Semi-quantitative PCR was used to measurechanges in c-myc mRNA extracted from cells treated for 30 min withAG1801 at 25 μM. GAPDH PCR was used as a control. AG1801 had minimaleffects on c-myc mRNA expression when assessed by this technique.

To determine the effect of AG and WP compounds on MM cellgrowth/survival MM cells were incubated with the indicated concentrationof AG or WP for 72 hours before cell growth and survival were estimatedby MTT assay. The results of this study are shown in FIGS. 3A-C.

An updated table summarizing additional data for other compounds of thepresent invention are included below in Table 1. The SAR for c-mycdownregulation and Stat3 inhibition as well as the IC50 values for eachcompound are shown below in Table 1.

TABLE 1 List of Kinase Inhibitors Tyrphostins-Biological Evaluation IC50IC50 IC50 (μM) (μM) (μM) V. STRUCTURE OCI MM1 U266 C-Myc

-Stat 3

AG  490 >12.5 >12.5 >12.5 ↓ >25 μM ↓

AG 1801   12.0    7.5    9.0 ↓ ↓

WP 1015 ND    1.9 ND ↓ ND

WP 1034    6.3    3.5    4.5 ↓ ND

WP 1038 >12.5    6.2   11.8 ↓ ↓

WP 1050 >12.5    2.1    5.0 ↓ ND

WP 1051 >12.5    5.5   11.1 ↓ ↓

WP 1065 ND    3.0 ND ND ND

WP 1066 ND    1.3 ND ↓ ND

WP 1073 ND  >2.5 ND ND ND

WP 1074 ND  >2.5 ND ND ND

WP 1075 ND  >2.5 ND ND ND

WP 1076 ND  >2.5 ND ND ND

WP 1077 ND    2.5 ND ND ND SYMBOLS: (more than) = > (less than) = < (notdone) = ND (inhibition = ↓ (no effect) = —

Two new compounds (WP1015 and WP1066) were synthesized based on previousSAR studies, as shown in the FIG. 4. These compounds were evaluated fortheir signal inhibitory and anti-proliferative/apoptotic properties onmultiple myeloma, lymphoma and chronic myelogenous leukemia cell lines.WP1015, WP1034, AG1801, and AG490 were tested across a range ofconcentrations (6,12, and 25 μM) for their ability to inhibit c-mycexpression using immunoblots. WP1015 was more active than previouslysynthesized WP compounds in inhibiting c-myc protein expression. AG490had little or no effect on c-myc expression at concentrations up to 50μM. Similarly, WP1015 was more effective in inhibiting Stat3phosphorylation in MM-1 cells than previous compounds.

WP1066 was then synthesized; the additional modification in WP1066(compared to WP1015) resulted in improved activity. As shown usingimmunoblots, WP1066 was more active in suppressing c-myc proteinexpression than WP1015. These immunoblots tested a range ofconcentrations (1.56-25 μM for WP compounds) and used β-actin as acontrol. Cells were also treated for 0-30 min. with the potenttranslation inhibitor, cycloheximide (CHX) to determine whether WP1066mediates similar effects on c-myc protein expression. CHX at much higherconcentrations did not result in the rapid reduction of c-myc as seen inWP1066 treated cells, suggesting possible effects on both translationand/or degradation of c-myc by WP1066.

Additional cell types were examined for response to treatment withWP1066. As shown using immunoblots, WP1066 caused rapid downregulationof c-myc protein in LP non-Hodgkin’ lymphoma cells as well as MM cells,demonstrating c-myc downregulatory activity is not restricted tomultiple myeloma cells alone. Multiple incubation periods for WP1066 (5,15, 30, 60 min.) were tested; a strong reduction in c-myc was observedat the shortest (5 min.) incubation period.

Further studies were conducted to determine the dose and time dependenteffects of these new compounds on IL-6 mediated Stat3 activation, c-mycprotein expression and anti-proliferative of LP and other cell types. Asshown in using immunoblots with a range (3-25 μM of WP compounds), bothWP1066 and WP1015 blocked IL-6 mediated Stat3 activation and reducedc-myc expression in LP cells. WP1066 had slightly better activity thanWP1015, demonstrating coincident improvement of compound action onmultiple cell types.

The anti-proliferative/apoptotic actions of WP1066 were also examined oncell lines known to overexpress c-myc. As shown in FIG. 5, WP1066treatment induced dose-dependent anti-tumor effects on multiple myeloma(MM-1), mantle cell lymphoma (Mino) and CML (WDT-2, WDT-3, K562, K562-R)cells lines, including those resistant to the kinase inhibitor Imatinibmesylate (K562-R). Thus WP1066 clearly exerts a potent inhibition ofcell proliferation and/or survival in multiple cancer cell lines, andWP1066 will be used as a therapeutic.

All of the compositions disclosed and claimed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and in the steps or in the sequence of steps of the methoddescribed herein without departing from the concept, spirit and scope ofthe invention. More specifically, it will be apparent that certainagents which are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 4 Compounds Display Potent Anti-Cancer Effects In Vivo

Compounds WP1129 and WP1130 were synthesized via the method described inExample 1. The structures of WP1129 and WP1130 are shown in FIG. 6. IC₅₀values for these compounds against MM-1 myeloma tumors are also shown inFIG. 6, and these compounds display IC₅₀ values even better thancompound WP1066. The increased potency of WP1129 and WP1130 support theuse of these compounds to treat cell proliferative diseases such ascancer.

An improved inhibition of c-myc/Stat3 by WP1066, WP1130, and WP1129 wasobserved (FIG. 7). The compounds were compared with regard to theirStat3/c-myc inhibitory activity in MM-1 cells. Strong inhibition ofc-myc/Stat3 was observed for WP1066, WP1130, and WP1129.

WP1066 was found to decrease tumor size in vivo. The results of animalstudies of human A375 melanoma tumors growing in nude mice, treated withWP1066 after tumors reached a palpable size, are shown in FIG. 8. Thefollowing animal model was used to evaluate the anti-tumor andanti-cancer effects of the compounds: on Day 0, A375 cells weresuspended to 20×106 cells/ml in RPMI 1640 medium. On day 0, 0.2 ml ofthis suspension containing A375 cells was injected (s.c.) into femaleSwiss nude mice 6-7 weeks of age. On Day 7, 40 mg/kg of WP1066 wasinjected (i.p.) into the above mice in a 0.1 ml suspension ofDMSO/PEG300 (50/50) on a qd, every other day schedule for 8 injections.Five mice per experimental group were used, including a vehicle(DMSO/PEG300) control group. Animals received 40 mg/kg WP1066 everyother day (QID) for a total of 8 injections. The control group reachedmaximum tumor burden at day 21, and for this reason the experiment wasstopped. WP1066 displayed strong anti-cancer and anti-tumor effects invivo. These results indicate that WP1066, and other compounds describedherein, may be used to treat hyperproliferative diseases such as cancer.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method of treating a disease selected from thegroup consisting of rheumatoid arthritis, inflammatory bowel disease,osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas,vascular occlusion, restenosis, pre-neoplastic lesions, carcinoma insitu, oral leukoplakia, melanoma, non-small cell lung cancer, small celllung cancer, lung cancer, hepatocarcinoma, retinoblastoma, astrocytoma,glioblastoma, neuroblastoma, mesothelioma, lymphoma, head cancer, neckcancer, breast cancer, pancreatic cancer, renal cancer, bone cancer,testicular cancer, ovarian cancer, cervical cancer, gastrointestinalcancer, colon cancer, bladder cancer, blood cancer, brain cancer, skincancer, eye cancer, tongue cancer, and gum cancer, comprising theadministration, to a patient in need thereof, of a therapeuticallyacceptable amount of a compound comprising the chemical formula:

wherein R₀ is chosen from the group consisting of:

where X₁, X₂, X₃, and X₄, are each independently selected from the groupconsisting of hydrogen, halogen, OH, and NO₂; R₂ is selected from thegroup consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, halogen,hydrogen, OH, NO₂, thioether, amine, SH, and NH₂; R₃ is:

where Z is NH; and where X₅ and X₆ are each independently chosen fromthe group consisting of hydrogen and lower alkyl; and R₄ is CN.
 2. Themethod of claim 1, wherein R₂ is hydrogen.
 3. The method of claim 1,wherein R₀ is:


4. The method of claim 3, wherein X₁ is a halogen.
 5. The method ofclaim 4, wherein X₁ is Br.
 6. The method of claim 1, wherein saidcompound has the structural formula:


7. The method of claim 1, wherein said compound has the structuralformula:


8. The method of claim 1, wherein said compound has the structuralformula:


9. The method of claim 1, wherein said compound has the structuralformula:


10. The method of claim 1, wherein said compound has the structuralformula:


11. The method of claim 1, wherein said compound has the structuralformula:


12. The method of claim 1, wherein the first compound is comprised in apharmaceutically acceptable excipient, diluent, or vehicle.